DeSUMOylation of a Verticillium dahliae enolase facilitates virulence by derepressing the expression of the effector VdSCP8

The soil-borne fungus Verticillium dahliae, the most notorious plant pathogen of the Verticillium genus, causes vascular wilts in a wide variety of economically important crops. The molecular mechanism of V. dahliae pathogenesis remains largely elusive. Here, we identify a small ubiquitin-like modifier (SUMO)-specific protease (VdUlpB) from V. dahliae, and find that VdUlpB facilitates V. dahliae virulence by deconjugating SUMO from V. dahliae enolase (VdEno). We identify five lysine residues (K96, K254, K259, K313 and K434) that mediate VdEno SUMOylation, and SUMOylated VdEno preferentially localized in nucleus where it functions as a transcription repressor to inhibit the expression of an effector VdSCP8. Importantly, VdUlpB mediates deSUMOylation of VdEno facilitates its cytoplasmic distribution, which allows it to function as a glycolytic enzyme. Our study reveals a sophisticated pathogenic mechanism of VdUlpB-mediated enolase deSUMOylation, which fortifies glycolytic pathway for growth and contributes to V. dahliae virulence through derepressing the expression of an effector.

To obtain complementary plasmid pNEO-VdSCP8-HA, the VdSCP8 (VDAG-_08085) gene, including the native promoter and terminator together with the HA fragment were ligated into a Hind III/EcoR I-linearized pNEO binary vector. The primers used above are listed in Supplemental data 1. pNEO-VdSCP8-HA construct was transformed into the knockout mutant VdΔscp8 to produce the complemented strains VdΔscp8/SCP8. The transformants were selected on PDA medium with 40 μg/mL G418. All the strains used in this study are listed in Supplemental Table 3.

Protein purification
To generate the plasmid pET-VdUlpB CD , the catalytic domain of VdUlpB (VdUlpB CD , 387-780 aa) was cloned and ligated into a BamH I/Xho I-linearized pET28α vector. For the plasmid pET-VdUlpB CDm , the cysteine (C711) in the catalytic domain of VdUlpB was mutated into serine using the Fast Site-Directed Mutagenesis kit (TIANGEN, KM101). All the primers used above are listed in Supplemental data 1.
For 2-D protein separation, 800 g of total proteins was loaded onto IPG strips (18 cm, pH 4-7; Cytiva, Cat#17123301). Isoelectric focusing (IEF) was carried out and the strips were then put on the top of SDS-PAGE gel. After electrophoresis, the protein spots were detected and quantified using Image Master 2D Platinum software (Cytiva, V6.0) and the volume ratios of corresponding spots between V592 and Vd T-DNA were calculated from three biological replicates. Protein spots with a ratio higher than 2, p value <0.05 (unpaired Student's t-test) were considered significant and manually excised for mass spectrometry analysis.

Mass spectrometry analysis
To identify whether VdEno was SUMOylated, Matrix-Assisted Laser Ions score is -10*Log(P), where P is the probability that the observed match is a random event. Individual ions score > 26 indicate identity or extensive homology (p < 0.05).
Protein scores are derived from ions scores as a non-probabilistic basis for ranking protein hits.

EMSA
The purified conserved DNA binding domain of VdEno (VdEno BD ) was incubated with specific probes for 1 h at room temperature. The competition experiment was conducted by adding a 50-fold molar excess of cold probes or nonspecific probes into the reaction before adding the specific probes. The products were analyzed with a 4% native PAGE gel and transferred to a HybondTM-N + membrane (GE, RPN303B). The probes were amplified with the primer pairs listed in Supplemental data 1, and they were labeled with 32 P as described above.

Chromatin immunoprecipitation (ChIP)
Conidia and mycelia of V. dahliae were cultured in liquid Czapek-Dox medium for 3 days and treated with 1% formaldehyde for 10 min at room temperature, followed